In primer walking sequencing, a primer/template complex is extended with a polymerase and chain terminated to generate a nested set of fragments whence the sequence is read after electrophoresis and detection (radioactive or fluorescent). A second primer is then synthesised using the sequence information near to the end of the sequence obtained from the first primer. This second (xe2x80x98walkingxe2x80x99) primer is then used for sequencing the same template. Primer walking sequencing is more efficient in terms of generating less redundant sequence information than the alternative xe2x80x98shotgunxe2x80x99 approach.
Disadvantages of the Current Art
The main disadvantage with primer walking is the resynthesis of the walking primer after each round of sequencing. Studier (J Kieleczawa et al, Science 258, p1787, (1992)) and others (T Azhikina et al, PNAS, 90, p11460, (1993)) have proposed elegant schemes using modular primers constructed out of presynthesised sets to avoid de novo primer synthesis after each round of sequencing. None of these walking primer schemes, however, are suited to cycle sequencing.
Cycle sequencing requires primers that have annealing temperatures near to the optimal temperature for the polymerase enzyme used for the cycle sequencing. Primers between 18 and 24 residues long are generally used for cycle sequencing.
As the length of the primer increases, then the size of any presynthesised walking primer set needed in order to achieve a given probability of obtaining a perfect match within a given number of contiguous overlapping frames also increases. For primers between 18 and 24 residues long, the size of the presynthesised walking primer set required makes primer walking an impractical proposition.
Advantages of the Current Invention
The current invention allows the use of a manageably sized set of presynthesised walking primers for cycled sequencing.
The Current Invention
The invention provides a method for primer walking sequencing of a nucleic acid target, which method comprises performing a series of sequencing reactions, each involving hybridising a primer to the target and effecting chain extension/chain termination of the primer, wherein for each sequencing reaction there is used a primer selected from a presynthesised set of walking primers of which the annealing temperatures are raised and/or the annealing properties improved without increasing their sequence complexity.
The invention also provides a library of y oligonucleotides, where y is defined as from 2 to 20000, each oligonucleotide comprising n nucleotide residues N and x nucleotide analogue residues X wherein
i) n is defined as 8 or 9,
ii) x is defined as 3-5,
iii) each nucleotide analogue residue X is defined as: either capable of base pairing with two or more of A C G and T, or forming stronger base interactions than A C G T,
iv) the order of the nucleotide residues N and the nucleotide analogue residues X in the oligonucleotide is defined as: either random or the residue X at the 5xe2x80x2-end of the oligonucleotide chain.
A DNA sequence is herein considered as a series of contiguous overlapping identically sized frames of residues with each frame translated from the next by one residue.